Luciferase Detection Assay System

ABSTRACT

The invention relates to methods and kits for detecting enzyme activity using bioluminescence. In particular, it relates to a novel assay system with increased light yield for a sensitive and convenient detection of luciferase activity, such as luciferase reporter enzyme activity. Provided is a method of detecting luciferase activity in a sample, comprising incubating the sample in the presence of luciferin and ATP to allow the generation of a light signal, wherein said light signal is enhanced by performing the incubation in a reaction mixture comprising phosphate and ammonium ions, and measuring the light signal. The invention also relates to kits for use in such method.

The present application is a continuation of application Ser. No.11/433,791 filed on May 12, 2006, which claims priority from U.S.Provisional Application bearing Ser. No. 60/681,093 filed 13 May 2005,and European Patent Application bearing Serial No. EP 05076165.9 filed18 May 2005. The foregoing applications are hereby incorporated byreference herein.

BACKGROUND OF THE INVENTION

The invention relates to methods and kits for detecting enzyme activityusing bioluminescence. In particular, it relates to a novel assay systemwith increased light production for a sensitive and convenient detectionof luciferase activity.

Bioluminescence is a naturally occurring phenomenon that has beenutilized for a number of applications, particularly in molecular biologywhere the enzyme associated with it have been used as genetic reporters.Bioluminescence is nearly ideal for use as a genetic marker. Typicallythere is no endogenous luminescent activity in mammalian cells, whilethe experimentally introduced bioluminescence is nearly instantaneous,sensitive and quantitative. While numerous species exhibitbioluminescence, only a relative few have been characterized and cloned.Of these, only Firefly (Photinus pyralis) luciferase, Renilla luciferaseand Aequorin have had much utility. Studies of the molecular componentsin the mechanism of firefly luciferases producing bioluminescence haveshown that the substrate of the enzyme is firefly luciferin, apolyheterocyclic organic acid,D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylic acid(hereinafter referred to as “luciferin”).

Firefly luciferase is a monomeric 61 kD enzyme that catalyses theoxidation of luciferin in a two-step process, which yields light at 560nm. The first step involves the activation of the carboxylate group ofluciferin by acylation with the alpha-phosphate of ATP in the presenceof magnesium to produce luciferyl adenylate with the elimination ofinorganic pyrophosphate (PPi). In the second step, the luciferyladenylate is oxidized with molecular oxygen to yield AMP, carbon dioxideand oxyluciferin. The oxyluciferin is generated in an electronicallyexcited state. Upon transition to the ground state the oxyluciferinemits light.

The reaction scheme of the reaction hereinafter referred to a‘luciferin-luciferase reaction’ is as follows:

Luciferase has many characteristics that make it ideal for a reporterenzyme. Its activity is not dependent on any post-translationalmodification, making it immediately available for quantitation. Inaddition, the luminescence is very bright, having very high quantumefficiency as compared to many other bio- and chemiluminescentreactions.

When light emission is initiated by the addition of luciferase into areaction mixture containing ATP, Mg²⁺ (or an other divalent cation suchas Mn²⁺), and luciferin, where all components are near or at saturatingconcentrations, one observes a rapid increase in light intensityfollowed by a rapid decrease in the first few seconds to a low level ofsustained light intensity that may last hours. This rapid decrease inthe rate of reaction has been thought to be due to product inhibition.These conventional “flash” type assays using firefly luciferase resultin a flash of light, which decays rapidly with the addition ofsubstrates to the enzyme. Especially in automated (e.g. robotic) assayprocedures, this is a major problem as it dramatically reduces the timewindow in which a signal, if present, can be detected. Means forextending the duration of the light signal in the luciferase assay havebeen eagerly sought.

One approach, which achieved some popularity, to solving the problem ofthe kinetics of the luciferin-luciferase reaction and the associateddifficulty of precisely measuring light emitted during the flash, was touse various inhibitors of the enzyme, which were reported to prevent theflash from occurring or to prolong light production. One such agent isarsenate. Arsenate lowers flash height and tends to prolong the lightemission period. The decrease of the intensity of the light signal isconsiderable undesirable, in particular when microtiter plates orinstruments capable of reading out strips are used. In addition, the useof arsenate is not desirable from an environmental point of view.

U.S. Pat. No. 4,246,340 likewise proposes a method for prolonging thelight signal in a luciferin-luciferase assay based on the use ofinhibitors, such as analogs of D-luciferin. This prolongs the lightsignal from seconds to a few minutes.

The cofactor coenzyme A (CoA) has been reported to affect the pattern oflight emission in the luciferin-luciferase reaction. Airth et al.,Biochimica et Biophysica Acta, vol. 27 (1958) pp. 519-532, report that,when CoA is added to a firefly luciferin-firefly luciferase reactionmixture, there is no effect on the initial peak of light intensity butluminescence will continue at a higher level for a time period that isproportional to the total CoA added. Airth et al. have shown that thetotal light emitted is greater in the presence of CoA than in itsabsence. U.S. Pat. No. 5,283,179 also discloses the addition of coenzymeA (CoA) in the assay reagent to yield greater enzyme turnover and thusgreater luminescence intensity. This resulted in an increased lightoutput that is maintained for at least 60 seconds.

It has been reported that other sulfhydryl compounds contribute to thestability of luciferases during preparation and storage of the enzymes.U.S. Pat. No. 4,833,075 discloses that dithiothreitol (DTT) willmaintain luciferase activity at a level of 50% in an aged Photinuspyralis luciferase solution which, without the DTT, would have only 10%residual enzymatic activity compared to a freshly prepared luciferasesolution. U.S. Pat. No. 4,614,712 describes that, when bacterialluciferase has been inactivated by disulfide formation, enzyme activitymay be restored by addition of DTT, 6-mercaptoethanol (6-ME), or otherreducing agents.

U.S. Pat. No. 5,618,682 describes compositions and methods forincreasing the duration of detectable photon emission of aluciferin-luciferase reaction. To that end, the reaction mixturecontaining luciferase, luciferin, ATP, and cofactors required forluciferase catalytic activity is mixed with a composition containingadenosine monophosphate, a radical scavenger (DTT) and a chelating agent(EDTA).

U.S. Pat. No. 5,650,289 reports that the combined use of CoA and DTT inthe reaction mixture positively influences the kinetics of theluciferin-luciferase reaction. The half-life of the light signal, i.e.the time period after which 50% of the original light signal isobserved, was assessed at 300 to 500 seconds.

It is an object of the present invention to provide an alternativeluciferase detection assay system with improved light yield andextended-glow light emission for sensitive detection of fireflyluciferase reporter enzyme.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that this goal is met by performing theluciferin-luciferase reaction in the presence of phosphate anions andammonium cations. The invention therefore relates to a method fordetecting luciferase activity in a sample, comprising incubating thesample, or a part thereof, in the presence of luciferin and ATP and anyrequired cofactor (e.g. Mg²⁺ or Mn²⁺) to allow the generation of a lightsignal, wherein said incubation is performed in a reaction mixturecomprising phosphate and ammonium ions to enhance said light signal, andmeasuring the light signal.

As is shown herein, the combined presence of ammonium and phosphate inthe reaction mixture yields a strong light signal which is stable for arelatively long period of time. A half-life of the light signal rangingfrom 30 minutes up to 8 hours can be achieved.

An advantage of the present invention is that it provides improvementsin the kinetics of light production and the total light produced in theluciferin-luciferase reaction which render assaying for luciferasesimple and sensitive. For example, with the methods or test kits of thepresent invention, the assays do not require special procedures, such asrapid sample injection, or special equipment, such as sophisticatedluminometers, to measure the light emitted in the rapid flash ofconventional luciferin-luciferase reactions. Indeed, using the methodsand compositions or kits of the present invention, it is possible thatdevices, such as scintillation counters, that are already available inmost laboratories for other types of measurements, can be employed inassays requiring measurement of light produced in a luciferin-luciferasereaction. This significantly expands application of such assays inscience and technology by facilitating use of the assays in laboratoriesthat do not have luminometers but do have scintillation counters orother devices for measuring light production.

Another advantage of the methods or test kits of the present inventionis that due to the sustained high signal a large number of samples canbe prepared in microplates and assayed for luciferase activity. This isespecially useful in both batch wise and continuous processing of thesamples in High Throughput Screening (HTS) applications.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the reaction mixture contains relativelyhigh concentrations of both phosphate and ammonium ions. Generallyspeaking, it is observed that the higher the phosphate concentration thehigher the total light yield of the luciferin-luciferase reaction. Thetotal light yield or total luminescence refers to the area under thecurve representing the detectable light units as a function of time. Itis determined by the peak intensity (i.e. the initial light burst uponcombining enzyme and substrates) and the rate at which the intensitydecreases after the peak intensity is achieved.

The total phosphate concentration in the reaction mixture is preferablyat least 10 mM, preferably at least 25 mM, more preferably at least 60mM, most preferably at least 100 mM. For example, a phosphateconcentration of 125 mM, 150 mM, 200 mM, 500 mM or 1 M can be used. Asexemplified in the Examples below, phosphate concentrations up to 1.4 Mphosphate were tested, which yielded good results in terms of theinitial light signal and the duration of the light signal. The term“phosphate” as used herein refers a polyatomic ion or radical consistingof one phosphorus atom and four oxygen. In the ionic form, it carries a−3 formal charge, and is denoted PO4³⁻. In a biochemical setting, a freephosphate ion in solution is called inorganic phosphate. Inorganicphosphate is generally denoted P_(i). Suitable sources of phosphateinclude phosphoric acid (H₃PO₄) and its salts, such as alkali salts orearth alkali salts of PO₄ ³⁻, HPO₄ ²⁻ and H₂PO₄ ⁻.

The term “ammonium ion” as used herein refers to a cation of the generalformula NR₄ ⁺, wherein R can essentially be any group. Preferred Rgroups include aliphatic groups like linear or branched hydrocarbons,optionally substituted e.g. by a hydroxyl, and hydrogens. The four Rgroups which coordinate the nitrogen atom can be the same or different.Ammonium ions can be supplied to the reaction mixture by any compoundcapable of providing a NR₄ ⁺ ion in an aqueous solution. In oneembodiment, ammonium ions are provided by one or more ammonium (NH₄ ⁺)salts, such as (NH₄)₂SO₄, NH₄H₂PO₄ and (NH₄)₂HPO₄.NH₄H₂PO₄ and(NH₄)₂HPO₄ are very suitable since these salts provide both phosphateanions as well as ammonium cations. In a further embodiment, ammoniumions can be provided by protonated amines. An amine is an organicderivative of ammonia (NH₃). One, two or three alkyl groups may replacethe hydrogens of ammonia to give primary, secondary or tertiary amines,respectively. Like ammonia, the ammonia derivatives, the amines, areweak bases. This means that aqueous solutions of amines are alkaline ifone mixes an amine with water, the lone pair on nitrogen can abstract aproton from water generating an ammonium ion and a hydroxide ion.

According to the present invention, a final concentration of 1 Methanolamine is assumed to provide the aqueous reaction mixture with 1Mof ammonium ions. Thus, the concentration of ammonium ions in thereaction mixture is regarded to be identical to the concentration of thecompound (buffer, salt etc.) source of ammonium ions in the reactionmixture.

Regular aliphatic amines have about the same base strength, with pKb'sin the range of 3-4, and are slightly stronger bases than ammonia. Theincrease in basicity compared with ammonia can be attributed to thegreater stability of an alkylammonium ion, as for example, RCH₂NH₃ ⁺,compared with the ammonium ion, NH₄ ⁺. This greater stability arisesfrom the electron-donating effect of alkyl groups and the resultingpartial delocalization of the positive charge from nitrogen onto carbonin the alkylammonium ion. Aromatic amines are much weaker bases, withpKb's around 9-13. This can be explained by resonance delocalisation ofthe lone pair electrons into the aromatic π-system.

As is clear from the above, the present invention discloses that thecombined presence of phosphate and ammonium ions in theluciferin-luciferase reaction mixture enhances the total luminescence ofa luciferase detection system. It is to be noted (see e.g. Example 4herein below) that this beneficial effect on total light yield is notobserved in a system for ATP detection which, as in a luciferasedetection system, also employs the luciferin-luciferase reaction. In anATP assay the large amount of luciferase (as assay reagent) and the lowamount of ATP (present in the sample) in combination with largequantities of Tris-phosphate ions, give rise to a lower totalluminescence than with no or lower quantities of Tris-phosphate presentin the assay mixture. In contrast, increasing the amount ofTris-phosphate ion concentrations enhanced the light output of theluminescence reaction in a luciferase detection assay mixture,comprising large quantities of ATP (being an assay reagent) and a lowamount of luciferase (from the sample). It has been reported that theenzyme characteristics of firefly luciferase differ between assays setup for ATP- and luciferase detection, respectively. See for example Yeet al. in “A Practical Guide to Industrial Uses of ATP-Luminescence inRapid Microbiology” published in 1997 by Cara Technology Limited,Lingfield). The distinct effects of the combination of ammonium andphosphate ions on the luciferase-detection system and the ATP-detectionsystem as disclosed in the present invention are very likely related tothe different enzyme characteristics. In one embodiment, a method of theinvention comprises detecting luciferase activity in a sample,comprising incubating the sample in the presence of luciferin and anexcess (relative to luciferase) of ATP and a bivalent cation to allowthe generation of a light signal, wherein the total light yield of saidlight signal is enhanced by performing the incubation in a reactionmixture comprising ammonium ions and at least 20 mM phosphate ions andammonium ions, and measuring the light signal. Preferably, theconcentration of ATP in the luciferin/luciferase assay mixture is atleast 0.05 mM, preferably at least 0.1 mM, for example in the range of0.5-5 mM ATP.

In a method of the invention, the amine used is preferably protonated atthe pH of the reaction mixture, which typically ranges between pH 5 and9. This means that the pKb of the amine is preferably below the desiredpH of the reaction mixture. Table 1 below lists some examples of aminesand their pKb values. Primary and secondary alkyl- or alkanolamines,such as ethylamine, diethylamine, ethanolamine and diethanolamine,appear particularly useful in a method of the invention. However,certain tertiary amines like triethylamine can also be used.

Amine-based buffers are particularly useful in practicing the presentinvention. Examples of amine-based buffers include Tris(tris(hydroxymethyl)aminomethane andBis-Tris(bis(2-hydroxyethyl)imino]-tris(hydroxymethyl)methane).Furthermore, heterocyclic amines may be used. In a heterocyclic amine, afive- or six-atom ring contains one or more nitrogen atoms. An exampleis imidazole.

TABLE 1 The structure, pKb and pKa of various amines Amine Structure pKbpKa Ammonia NH₃ 4 74 9.26 Primary Amines methylamine CH₃NH₂ 3 36 10.64ethylamine CH₃CH₂NH₂ 3 19 10.81 isopropylamine (CH₃)₂CHNH₂ 3 18 10.82tert-butylamine (CH₃)₃CNH₂ 3 17 10.83 cycloheylamine C₆H₁₁NH₂ 3 34 10.66Secondary Amines dimethylamine (CH₃)₂NH 3 27 10.73 diethylamine(CH₃CH₂)₂NH 3 02 10.98 piperidine

3 25 10.75 Tertiary Amines trimethylamine (CH₃)₃N 4.19 9.81triethylamine (CH₃CH₂)₃N 3.25 10.75 Aromatic Amines aniline

9.37 4.63 4-methylaninline

8.92 5.08 4-chloroaniline

9.85 4.15 4-nitroaniline

13.0  1.0 Heterocyclic Aromatic Amines pyridine

7.75 5.25 imidazole

7.05 6.95Still further, zwitterionic compounds can be used to provide theluciferase reaction mixture with ammonium ions. By definition, azwitterion (from the German “zwei”, meaning “two”) is an ion thatcontains both positively and negatively charged ions in the samemolecule. A zwitterion can be formed by compounds which contain both,acidic and basic groups. Amino acids are typical examples ofzwitterionic compounds. Since both carboxyl and amino functions arepresent in amino acids, these two functional groups tend to react witheach other in the same way, yielding an “internal salt”, which containsboth the carboxylate anion and an ammonium cation function. Theequilibrium lies far to the salt, so the predominant structure of anamino acid is the zwitterionic structure. For example, glycine containsboth a basic amino group (NH₂) and an acidic carboxyl group (COOH); whenthese are both ionized in aqueous solution, the acid group loses aproton to the amino group, and the molecule is positively charged at oneend and negatively charged at the other. In a substantially acidicsolution, the carboxylic acid function of the zwitterion is protonated,thus forming a cationic structure which has an ammonium ion functionalgroup. This is the conjugate acid of the zwitterion (and also of theneutral, uncharged, form of the amino acid, by protonating the aminogroup). According to the invention, zwitterionic compounds having a pKaof the conjugated acid of the amino group above the pH of theluciferine-luciferase reaction mixture are preferred, as this ensuresthat the amine moiety is protonated.

Another class of useful zwitterionic compounds is represented by thezwitterionic buffers. Table 2 lists some exemplary common zwitterionicbuffers which may be used in the present invention. The invention ishowever not limited to the use of those zwitterionic buffers listedbelow.

TABLE 2 Zwitterionic Buffers pKa at useful 20° C. pH Buffer Structureand Name MW (D/° C.) range ACES

N-(2-acetamido)-2-aminoethanesufonic acid 182.2 6.88(−0.020) 6.4-7.4 ADA

N-(2-acetamido)iminodiacetic acid 190.2 6.62(−0.011) 6.4-7.4 BESHO₃SCH₂CH₂N(CH₂CO₂H)₂ 213.3 7.17 6.6-7.6 N,N-bis(2-hydroxyethyl)-2-(−0.016) aminoethanesulfonic acid Bicine HO₂CCH₂N(CH₂CO₂H)₂ 163.2 8.357.8-8.8 N,N-bis(2-hydroxyethyl)glycine (−0.018) CAPS C₆H₁₁NH(CH₂)₃SO₃H221.3 10.4  9.7-11.1 3-(cyclohexylamino)-1- (0.032) propanesulfonic acidCHES C₆H₁₁NH(CH₂)₂SO₃H 207.1 9.55  9.0-10.1 2-(cyclohexylamino)-1-(−0.011) ethanesulfonic acid HEPES

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid 238.3 7.55(−0.014)7.0-8.0 HEPPS(EPPS)

4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid 252.3 8.0(−0.011)7.6-8.6 MES

2-morpholinoethanesulfonicacid monohydrate 213.3 6.15(−0.011) 5.8-6.5MOPS

3-morpholinopropanesulfonicacid 209.3 7.2(−0.011 6.5-7.9 PIPES

piperazine-1,4-bis(2-ethanesulfonic acid) 302.4 6.82(−0.009) 6.4-7.2TAPS (HOH₂C)₃CNH(CH₂)₃SO₃H 243.3 8.4 7.7-9.1N-[tris(hydroxymethyl)methyl]-3- (0.018) aminopropanesulfonic acid TES(HOH₂C)₃CNH(CH₂)₂SO₃H 229.3 7.5 7.0-8.0 N-[tris(hydroxymethyl)methyl]-2-(−0.020) aminoethanesulfonic acid Tricine (HOH₂C)₃CNHCH₂CO₂H 179.2 8.157.6-8.8 N-[tris(hydroxymethyl)methyl]- (−0.021) glycineIn a method of the invention, the reaction mixture pH typically rangesbetween 5 and 9, preferably between 6.5 and 8.2.

As will be clear from the above, many combinations of phosphate sourcesand ammonium sources can be used in a luciferase reaction mixture of theinvention. More than one source for a given ion may also be used.Certain combinations of phosphate/ammonium sources were found to beparticularly useful. Of specific interest for use in the presentinvention is the combination of an amine-based ionic buffer, like Trisor Bis-Tris, which is neutralized with phosphoric acid.

The concentration of ammonium ions in the luciferase reaction mixture ofthe invention can vary. A concentration of at least 20 mM, better morethan 60 mM ammonium ions is preferred. Very good light yields can beobserved at a concentration of more than 100 mM ammonium ions, evenbetter at more than 200 mM ammonium ions, for example 250 mM Tris orethanolamine or 500 mM ammonium (NH₄ ⁺).

According to the invention, the concentration of phosphate ions andammonium ions in the reaction mixture can be the same or they can bedifferent. In one embodiment, the concentration of ammonium ions ishigher than the concentration of phosphate ions. In another embodiment,the concentration of ammonium ions is lower than the concentration ofphosphate ions. In a specific aspect, ammonium ions are present inexcess of the phosphate ions, for example at least a 1.1-fold excess,preferably at least 1.4-fold excess, such as 1.5, 1.7, 1.8 or even2-fold excess of ammonium ions. As exemplified below, very good resultscan be obtained using Tris as source of ammonium ions in excess (e.g.2-fold) of phosphoric acid (as source of phosphate ions), in particularwith a phosphate concentration of at least 60 mM and/or with an ammoniumion concentration of at least 120 mM (see Table 3). See Tables 4 and 5which demonstrate the effect of increasing concentrations ethanolamine(Table 4) or ammonium (Table 5) and phosphate on the total light yieldof the luciferase reaction. Also here, ammonium ions are present inexcess of phosphate ions. This is the result of neutralizing thesolution containing the ammonium ions (Tris, ethanolamine,ammoniumphosphate) with phosphoric acid to pH 7.0. Of course, it is nota prerequisite that the final pH of the aqueous reaction mix iscontrolled solely by the ammonium and phosphate ions. For example, theinvention also encompasses a reaction mixture comprising phosphate ionsin excess of ammonium ions which is neutralized by an alkaline agentwhich is not a source of ammonium ions, like NaOH, KOH and the like. Ina similar fashion, acidic agents other than those contributing phosphateions can be used to set the pH at a desired value.

In one embodiment, the combination of Tris buffer and phosphoric acid isused. For example, a two-fold detection buffer comprising 125 mM Trisneutralized with 63 mM H₃PO₄ provides sufficient phosphate and ammoniumions to the reaction mixture to positively affect the magnitude andkinetics of the light signal. Increasing the Tris and/or phosphateconcentration further enhances this effect (see also FIG. 1). Verysatisfactorily results were obtained in a reaction mixture comprising atleast 250 mM Tris, accompanied with at least 125 mM phosphate.

Other useful combinations include mono- and di-alkanolamines andphosphoric acid. In one aspect, the luciferase reaction mixturecomprises ethanolamine neutralized with phosphoric acid to approximatelypH=7.0 (see FIG. 2).

The reaction mixture preferably also comprises a stabilizer forluciferase. Suitable examples of stabilizers are mammalian serumalbumin, a lactalbumin and an ovalbumin. The use of bovine serum albumin(BSA) is preferred. The amount of stabilizer will typically be below 1wt %, calculated based on the total weight of the reaction mixture.Further, the reaction mixture preferably comprises a sequestering agentlike ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid (EGTA) orcyclohexane-1,2-diaminetetraacetic acid (CDTA). The sequestering agentis generally present in an amount of 0.1 to 25 mM.

Methods for detecting luciferase activity known in the art typicallycomprise thiol-containing compounds, such as DTT and/or CoA (see forexample U.S. Pat. No. 5,650,289). Whereas thiols reportedly contributeto the stability of luciferase and improve the kinetics of lightproduction, they have several drawbacks. Formulations containing thiolshave a pungent odour. Furthermore, thiols experience auto-oxidation insolution and, as a consequence, are not stable for long times. Thepresent inventors unexpectedly observed that thiol-compounds in aluciferase reaction mixture are no longer necessary if both ammonium andphosphate ions are present. It was found that the overall lightproduction in the absence of thiols but in the presence of ammonium ions(e.g. provided by a Tris buffer) and phosphate ions was similar to oreven surpassed the light production in the presence of thiols. Herewith,the invention provides a method of detecting luciferase activity in asample, comprising incubating the sample in the presence of luciferin,ATP and a divalent cation (e.g. Mg²⁺ or Mn²⁺) to allow the generation ofa light signal, wherein said incubation is performed in a reactionmixture comprising phosphate and ammonium ions in the relative absenceof a thiol-containing compound, and measuring the light signal. Theexpression “in the relative absence of a thiol-containing compound” ismeant to indicate that no exogenous or supplementary thiol compound,such as DTT, glutathione and/or CoA, is added to the reaction mixture.In one embodiment, the reaction mixture and assay reagent of theinvention is free or essentially free of thiol-compounds. It may howeveroccur that a sample to be assayed for luciferase activity contributestrace amounts of thiol-compounds, e.g. thiols of cellular origin, orfrom a separate lysis solution to the reaction mixture. In a preferredembodiment, the invention provides a method of detecting luciferaseactivity in a sample, comprising incubating the sample in the presenceof luciferin and ATP and a bivalent cation to allow the generation of alight signal, wherein the total light yield of said light signal isenhanced by performing the incubation in a reaction mixture comprisingammonium ions and phosphate ions but no exogenous or supplementary thiolcompound (i.e. no thiol-compounds of non-cellular origin), and measuringthe light signal. Preferably, the reaction mixture comprises at least 20mM, more preferably more than 60 mM, like 100, 150, 200, 250, 350, 400,500 mM or even 1 or 1.5 M phosphate ions, optionally in combination withat least 60 mM or preferably at least more than 100 mM ammonium ions.

Whereas the teaching of the invention makes the inclusion ofthiol-compounds unnecessary, it is to be noted that the presentinvention is not limited to assay conditions without thiol-compounds.DTT, glutathione and/or CoA, or any other commonly used assay componentknown in the art, can be present in the reaction mixture. However, theuse of an odourless, non-thiol reducing agent is preferred. For example,a water-soluble, organic phosphine-containing compound such as Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) may be used (seeWO03/044223) in a method of the invention. The present inventors alsoidentified other suitable reducing agents as being suitable for use inluciferin-luciferase reaction mixture, such as thiosulfate, sulfite anddithionite (see Example 3 and FIG. 4). The reducing agent is typicallypresent in an amount of 0.1 to 10 mM.

Apart from the substances mentioned above, the reaction mixture maycontain all usual substances which are used in luciferin-luciferasereaction mixtures.

A method of the invention allows the sensitive and convenient detectionof luciferase activity in a sample. A sample may comprise cells(suspected of) comprising luciferase activity. A sample can be asuspension, an extract or lysate of cells, for example cells that havebeen provided with a nucleic acid encoding luciferase. Beyond theavailability of crystalline luciferases isolated directly from the lightorgans of beetles, cDNA's encoding luciferases of several beetle species(including, among others, the luciferase of P. pyralis (firefly), thefour luciferase isozymes of P. plagiophthalamus (click beetle), theluciferase of L. cruciata (firefly) and the luciferase of L. lateralis)(de Wet et al., Molec. Cell. Biol. 7, 725-737 (1987); Masuda et al.,Gene 77, 265-270 (1989); Wood et al., Science 244, 700-702 (1989); EP 0353 464) are available. Further, the cDNA's encoding luciferases of anyother beetle species, which make luciferases, are readily obtainable bythe skilled person using known techniques (de Wet et al. Meth. Enzymol.133, 3-14 (1986); Wood et al., Science 244, 700-702 (1989). Alsoencompassed is the detection of mutant luciferase activity, for exampleluciferase that is genetically engineered to improve its properties,such as a non-naturally occurring luciferase with an increased pHstability or increased thermal stability. Other useful mutants areluciferases which include modifications which cause a change in color inthe luminescence that is produced. These are for example described inU.S. Pat. No. 6,387,675.

Firefly luciferase can be reliably expressed from various expressionvectors and in a diversity of organisms as a reporter in studies of generegulation. Luciferase reporter assay systems are currently one of thebest non-toxic, rapid and sensitive methods to measure gene expression.The assay is based on the detection of luciferase activity whichcorrelates with transcription due to DNA regulatory elements in genes,mutations within those elements as well as responses to extracellularand intracellular signals.

In a preferred embodiment, a method of the invention is applied toquantify the activity of luciferase that is used as reporter enzyme. Thebasic steps for the use of the luciferase reporter gene system are asfollows: 1) construction of an appropriate luciferase reporter vector;2) transfection of the plasmid DNA into cells to allow luciferaseexpression; 3) optionally preparation of cell extracts and 4)measurement of luciferase activity evidenced by a light signal.According to the invention, the latter measurement is performed in thepresence of both phosphate and ammonium to increase the overall lightyield.

As with all reactions, temperature affects the rate of luciferaseactivity. An accepted range for optimal luciferase assay is between20-25° C. Ambient room temperature falls within this range and isconvenient for performing the assay. Samples and all buffers aretherefore preferably allowed to equilibrate to room temperature toobtain consistent and reproducible results. At temperatures above 30°C., the light emission shifts to red and the enzyme quickly degrades.

Under conditions of excess ATP, luciferin and Mg²⁺ relative to theamounty of luciferase to be detected, the initial peak height andintegrated total light output from a reaction is proportional to theamount of functional luciferase enzyme. The light production optimumhowever occurs only in rather narrow substrate concentration rangeswhich exceed the Km value. At lower ATP concentrations, light emissionpeak heights are reached and remain at that level for extended periodsof time while at higher ATP concentrations the initial light burst isincreased but so is the rate of decay. There exist two sources of ATP inluciferase lysate: the known amount which is added to the sample and theunknown and variable amount of endogenous cellular ATP which isco-extracted in the luciferase sample. The absolute final concentrationwill be somewhat uncertain. For most assay conditions, 0.5-2.5 mM ATP inthe assay reaction mixture will be optimal.

The remaining reaction cofactor and substrate components of the system,primarily Mg²⁺ (or Mn²⁺) and luciferin, also have optimalconcentrations. They tend to have a wider range of these values thanthat of ATP. Magnesium ions are preferably present in an amount of atleast 0.5 mM. Usually, the concentration of Mg²⁺ in the reaction mixturedoes not exceed 50 mM. For most conditions, a final luciferinconcentration of 0.5-1.0 mM and Mg²⁺ concentrations of 1-5 mM are nearoptimum. The relatively low concentrations of Mg²⁺ in cell extracts aregenerally insufficient to alter reaction rates and firefly luciferin isnot a natural component found in other organisms. The use of Mg²⁺ andCa²⁺ free wash buffers for cells is recommended if cell lysates areprepared. Luciferin is preferably present in an amount of between 0.01and 3 mM, more preferably between 0.05 and 1.0 mM.

When relative experimental luciferase activities are to be correlated toan absolute value, a purified luciferase standard curve can be utilized.Since measured levels of activity are normally stated in RLUs, orrelative light units, an absolute value is not being determined. RLUvalues can vary significantly with the same sample when measured bydifferent instruments due to variations in equipment design andcalibration. A specific activity certified luciferase standard, whenreconstituted to the manufacturer's specifications, will result in asignal derived from a known luciferase quantity. These known specificactivity reference values may be used to normalize arbitrary RLU samplemeasurements to a given instrument. Additionally, a purified luciferasestandard does not necessarily represent the exact amount of luciferaseproduced by transfected cells, since the specific activity of theexpressed luciferase may differ from the purified luciferase.

For the measurement of (heterologously) expressed luciferase activity ina method of the invention, a suspension of cells suspected to containluciferase can simply be added to a reaction buffer comprising a lysisagent. No separate lysis step is thus required. Upon lysis of the cells,the luciferase becomes accessible to other components of the reactionbuffer (substrates, cofactors, ammonium and phosphate ions, and thelike) such that the luciferin-luciferase reaction can take place.Typical lysis agents are non-ionic detergents such as Tergitol™ NP-9,Triton™ X-100, Thesit™ and Tween™ 20.

In another embodiment, the sample comprises a cell lysate or cellextract, i.e. the luciferase is first extracted from cells expressingluciferase. There are two main approaches for extraction of luciferase,either through mechanical or chemical methods. Both methods haveadvantages and disadvantages and evaluation of the method used isimportant for efficient luciferase extraction and optimal assaysensitivity.

A cell lysate comprises cellular components that are no longer organizedinto a recognizable intact cellular architecture. Cell lysates may havesoluble and insoluble components, either of which may be removed beforeusing the lysate. Lysates may be prepared by any means, includingmechanical disruption using sonication, a dounce, mortar and pestle,freeze-thaw cycling, or any other device or process that destroys thephysical integrity of cells; or chemical lysis by detergents such aszwitterionic and nonionic detergents, or cationic detergents The mostcommonly used mechanical lysis method for cell disruption is sonication.Due to large amounts of energy coupled into the sample, heating candevelop. Luciferase is heat labile; therefore the sonication methodshould take this into account by using short sonication cycles. Samplecooling along with five 5-10 second sonication bursts is normallyadequate for lysis with minimal luciferase inactivation. Freeze-thawlysis with multiple cycles is also widely utilized for luciferaseassays. The disadvantage of this mechanical lysis method is thatluciferase tends to stay bound to the cellular debris pellet aftercentrifugation. An advantage is the lack of interference from celldisruption chemicals.

Significant improvement and convenience of lysis of eukaryotic cells canbe achieved by chemical lysis. Using non-ionic detergents such as 1%Triton™ X-100, greatly enhances solubilization of luciferase fromcellular components. Due to better recovery and luciferase activitystimulation by detergent, as much as a 25-fold greater light output ispossible over simple freeze-thaw lysis. Other cationic and zwitterionicdetergents can be useful for eukaryotic cell lysis and tend to stimulateluciferase activity as well. Anionic detergents, however, generallyinhibit luciferase activity. A proper buffering system is also requiredin optimizing enzyme activity. Lysis reagents with Tris-phosphate,Tricine or phosphate buffers, pH 7.5 to 8.0, are preferred for maximalluciferase stability. The addition of DTT can aid in preserving enzymefunction; chelating agents like EDTA or EGTA are preferably included forchelation of e.g. Mg²⁺ and Ca²⁺-ions to suppress the activity of e.g.proteases that might adversely affect the luciferase and chelation ofheavy metal ions which may inhibit luciferase activity. Due to thenature of bacterial physiology, the inclusion of detergents alone forbacterial lysis proves inadequate. Additional disruption of thebacterial cell walls generally requires enzymatic and or mechanicallysis steps in order to be successful. The combination of 1% TritonX-100 and 1 mg/ml lysozyme can be used for lysis without affectingluciferase activity or stability. Moderate amounts of luciferase boundto cell debris remain unextractable. A possible alternative is the useof unclarified bacterial lysate without centrifugation, which mayincrease sensitivity but lower reproducibility because ofnon-homogeneous distribution of enzyme in the extract.

Another aspect of the invention relates to an assay reagent for thedetection of luciferase activity which comprises phosphate and ammoniumions. Said assay reagent may be in a concentrated form which upondilution into the reaction mixture provides the desired finalconcentrations of phosphate and ammonium ions. Preferred (concentrated)assay reagents comprise at least 40 mM phosphate, more preferably atleast 100 mM phosphate, most preferably at least 250 mM phosphate ionsand/or at least 20 mM ammonium ions, preferably at least 100 mM, mostpreferably at least 250 mM ammonium ions. In a specific aspect, theammonium ions in the assay reagent are present in excess of phosphateions, for example 1.1-fold excess, or 1.5-2.0-fold excess or even higherexcess. Preferred assay reagents are those containing Tris, ethanolamineand/or ammonium phosphate (NH₄)₂HPO₄ neutralized with phosphoric acid toneutral pH. However, other combinations of ammonium and phosphatesources may be used as well. Exemplary assay reagents are thosedescribed in the Examples below.

The assay reagent may be in a liquid form (e.g. an aqueous solution) orin a solid form which upon reconstitution provides the phosphate andammonium ions. Preferred assay reagents comprise as a source of ammoniumions a compound selected from the group consisting of ammonium salts, anamine, a zwitterionic compound, and combinations thereof. Otherpreferred assay reagents comprise as a source of phosphate phosphoricacid or a salt thereof. An assay reagent may also comprise other usefulsubstances, such as a stabilizer, chelating agent, reducing agent and/orcell lysing agent. In one embodiment, the invention provides an assayreagent comprising all the components necessary for aluciferin-luciferase reaction to occur, except for the luciferase. Thistype of “all-but-one” assay reagent can simply be contacted with asource of luciferase, e.g. a suspension of cells expressing luciferase,to obtain a light signal. An example of an “all-but-one” assay reagentcomprises a Tris-phosphate buffer pH 7, D-luciferin, ATP, Mg²⁺, EDTA,Ca²⁺, thiosulfate and a non-ionic detergent to lyse the cells. A personskilled in the art will be able to prepare many variants of the assayreagent, as long as both ammonium and phosphate ions are present.

In a preferred embodiment, the assay reagent does not comprise DTTbecause luciferin was found to be more stable in the absence of DTT. Thestability of luciferin in the absence of DTT allows to prepare anall-but-one assay reagent with a sufficiently long shelf-life.

Also provided is a kit for use in a luciferase activity assay, forinstance a gene reporter assay, wherein said kit comprises componentsrequired to perform a luciferase activity assay as provided herein. Suchkit components include luciferase substrate (luciferin), cosubstrate(ATP), cofactors such as Mg²⁺ or Mn²⁺, a reducing agent such as sodiumthiosulfate and a source of phosphate and ammonium ions.

Preferably, the kit comprises an assay reagent mentioned above. Otheruseful kit components include a lysis buffer, preferably a non-ionicdetergent. Preferably, the kit components are put together in a minimalnumber of separate containers. Premixing of components reduces thenumber of handling steps performed by the user of the kit. In oneembodiment, the invention provides a luciferase detection kit comprisinga first container with a substrate mixture comprising luciferin, ATP anda reducing agent and a second container with a buffer solutioncomprising at least phosphate and ammonium ions and a bivalent cation.The buffer solution may furthermore comprise further useful substances,such as a stabilizer, chelating agent and/or cell lysing agent Ofcourse, either or both the substrate mixture and the buffer solution maycomprise other useful ingredients, such as additional (buffer) salts,chelating agents, cell lysing agent, bivalent cations (Mg²⁺; Mn²⁺),stabilizers and the like. In a preferred embodiment, a kit comprises alyophilised substrate mixture that can be reconstituted with the buffersolution to yield a ready-to-use detection buffer. The freshly prepareddetection buffer can then be added to a sample to be analysed forluciferase activity. Any of the phosphate and ammonium ion sourcesmentioned above may be used in the buffer solution. In a specificaspect, the invention provides a kit wherein the ammonium ion in buffersolution is provided by Tris or (di)ethanolamine. Said buffer solutionis preferably neutralized with H₃PO₄.

A kit of the invention may also comprise a cell lysing solution. A kitmay furthermore comprise (purified) luciferase enzyme which can be usedas a positive control or to prepare a luciferase standard curve.

In a specific aspect, a kit of the invention comprises a freeze-driedsubstrate mixture and a buffer solution which is to be added to thefreeze-dried substrate mixture to yield a ready-to-use luciferasedetection reagent. The freeze-dried substrate mixture comprises ATP,luciferin and thiosulfate. The substrate mixture may further compriseother useful components known in the art, such as AMP which furtherenhances the half-life of the light-signal. The buffer solutioncomprises ammonium, phosphate, detergent (cell lysing agent), Mg²⁺,EDTA, Ca²⁺ and (NH₄)₂SO₄.

The invention is illustrated by the Examples below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Light-output time course of the luciferin-luciferase reaction atdifferent concentrations of ammonium ions (Tris) and phosphate ions.

FIG. 2: Light-output time course of the luciferin-luciferase reaction atdifferent concentrations of ethanolamine and phosphate (panel A) orammonium and phosphate (panel B).

FIG. 3: Light-output time course of the luciferin-luciferase reactioncomprising Tris (0.5 M), phosphate (0.25 M) and various reducing agents.

FIG. 4: Effect of different Tris-phosphate concentrations in thereaction mixture on reaction kinetics of a luminescence reaction in(panel A) an ATP-detection system with small amounts of ATP (5 nM) andlarger quantities of luciferase (5 mg/l≈8.3×10⁻⁸ M) or (Panel B) aluciferase-detection system with small amounts of luciferase (5.0×10⁻⁵mg/l≈8.3×10⁻¹³ M) and excess (1.5 mM) ATP.

FIG. 5: Total light yield of each luminescence reaction of the ATP andluciferase assay systems of FIGS. 4A en 4B. The integral of eachreaction kinetics curve with different Tris-phosphate ion concentrationwas determined between 1 and 420 minutes. The resulting totalluminescence is expressed in MegaCounts (10⁶ counts) as a function ofphosphate ion concentration.

FIG. 6: Effect of phosphate concentration on the half-life (calculatedafter 10 minutes of incubation) of the luminescence reaction of the ATPand luciferase assay systems of FIGS. 4A en 4B. The half-life is thetime needed for the luminescence to decay to half of its initial value.

EXAMPLES Example 1 Effect of Different Concentrations of Ammonium andPhosphate Ions

This example demonstrates the effect of different concentrations of Trisand phosphate on the performance of a luciferin-luciferase reaction.

A stock solution of 4.0 M Tris (source of ammonium ions) was neutralizedwith phosphoric acid (H₃PO₄) until a pH of 7.0 was reached. This stockbuffer solution contained 2.0 M of phosphate. The stock solution wasused to make a range of assay reagents comprising, respectively, 2.2,1.1, 0.55, 0.275, 0.138, 0.069, 0.034 and 0.017 M Tris. The phosphateconcentration in these assay reagents was 1.1, 0.55, 0.275, 0.138,0.069, 0.034, 0.017 and 0.085 M phosphate, respectively.

Each reagent was checked for its pH to be 7.0 and was adjusted withsmall amounts of H₃PO₄ (several μl of a 2 M H₃PO₄ solution) if needed.The phosphate anions added in this manner had no significant effect onthe above reported phosphate concentration. Each assay reagent wassupplemented with D-luciferin, ATP and sodium-thiosulfate (Na₂S₂O₃) bythe addition of 250 μl of a freshly prepared stock solution (30 mM ATP,3 mM D-luciferin, 100 mM thiosulfate adjusted to pH=7.0 with 1 M NaOH)to 2250 μl of assay reagent.

Luciferase was lyophilized in Dulbecco's-PBS with Mg and Ca (D′PBS⁺⁺)containing 0.05 g/ml trehalose and 0.016 g/ml BSA). Luciferase sampleswere prepared by reconstituting lyophilized luciferase in 200 μl water.The resulting solution was then diluted 100-fold by adding 150 μl of thereconstituted luciferase to 15 ml PBS (8.1 mM NaH₂PO₄, 1.5 mM KH₂PO₄,2.7 mM KCl and 137 mM NaCl) containing 5 mM MgCl₂, 5 mM CaCl₂, 5 mMEDTA, 0.4% Thesit (a non-ionic detergent) and 0.1% bovine serum albumin(BSA). This yielded the “luciferase sample” solution which contained 27ng/ml of luciferase.

To measure the luminescence kinetics of the luciferin-luciferasereaction for each Tris-phosphate concentration, 100 μl of each assayreagent (Tris-phosphate dilution with D-luciferin, ATP and thiosulfate)was added to a well of a white 96-wells microplate after which 100 μlsample solution was added. The microplate was shaken for 10 seconds at1100 rpm on a microplate-shaker and then immediately placed in aTopCount®-NXT Microplate Scintillation and Luminescence Counter. Theresulting luminescent light signal was measured immediately afterloading the microplate into the counter. The measurement was repeatedafter 5 and 10 minutes. Hereafter the signal was measured repeatedlywith 15 minute intervals to a total time of 420 minutes. Between thesuccessive measurements the microplate was kept constantly in thecounter at 22° C. The emitted light was quantified as counts per second(cps), count time 3 seconds per well.

Final Tris concentrations in the different reaction mixtures were: 1.0,0.5, 0.25, 0.125, 0.063, 0.031, 0.016 and 0.008 M. The phosphateconcentrations were 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 and0.004 M phosphate ions respectively. Final Mg²⁺, D-luciferin, ATP,thiosulfate and luciferase concentrations in the reaction mixtures were2.5 mM, 150 μM, 1.5 mM, 5 mM and 14 pg/μl, respectively.

FIG. 1 illustrates the effect of increasing Tris-phosphateconcentrations in the luciferin-luciferase reaction mixture on theinitial height and the decay of the light signal. To compare the totallight yield (expressed as 10⁹ counts) of each reaction mixture the areasunder the curves were calculated by integrating the equations of thecurves between 0 and 420 minutes. These equations of the curves wereobtained using the option ‘trend line with equation’ of Microsoft Excel®software. The results of these light yield calculations are summarizedin Table 3.

FIG. 1 and Table 3 show that increasing the ammonium ions and phosphateions in luciferase-detection reaction mixture result in both an increaseof the initial light signal as well as total light yield up to a finalconcentration of 0.5 M Tris/0.25 M phosphate. Above these concentrationsthe initial light signal and, to a smaller extent, the total light yielddecreased relative to the values obtained using 0.5 M Tris/0.25 Mphosphate, indicating that these concentrations are above the optimalconcentrations. As is clear from Table 3, the use of supra-optimalconcentrations of ammonium and phosphate ions still result in improvedlight yield when compared to low or very low ammonium/phosphateconcentrations. Therefore, a method of the invention is not limited tothe use of suboptimal or optimal ion concentrations.

TABLE 3 Light yield of the luciferin-luciferase reaction as function ofTris and phosphate concentration from FIG. 1 [Tris] [Phosphate] LightYield (M) (M) (10⁹counts) 1.0 0.5 123.8 0.5 0.25 137.3 0.25 0.125 126.80.125 0.063 103.4 0.063 0.031 80.4 0.031 0.016 63.0 0.016 0.008 52.90.008 0.004 44.4

Example 2 Effect of Different Sources of Ammonium Ions

This example demonstrates the advantageous effects on light yield ifethanolamine or ammonium phosphate is used to provide theluciferin-luciferase reaction with ammonium ions.

Stock solutions of 4.0 M ethanolamine (H₂NCH₂CH₂OH) and 3.0 Mammonium-phosphate ((NH₄)₂HPO₄) were neutralized with phosphoric acid(H₃PO₄) until pH 7.0 was reached. Dilutions were made in the same manneras described in example 1. Furthermore, all methods and other chemicalsused to create the assay mixture and to test this for its light yield ina luciferin-luciferase reaction, were standardized and thus the same asin example 1. Final ethanolamine concentrations in the assay were 1.0,0.5, 0.25, 0.125, 0.063, 0.031, 0.016 and 0.008 M, and 0.5, 0.25, 0.125,0.063, 0.031, 0.016, 0.008 and 0.004 M phosphate, respectively.According to the present invention, a final concentration of 1Methanolamine is assumed to provide the aqueous, neutral reaction mixturewith 1M of ammonium ions. Thus, the concentration of ammonium ions inthe reaction mixture is regarded to be identical to that of the sourceof ammonium ions.

In the case of (NH₄)₂HPO₄, the ammonium salt provides both the ammoniumand phosphate ions. In the dilutions tested, the ammonium (NH₄ ⁺)concentrations in the final mixture were 2.0, 1.0, 0.5, 0.25, 0.125,0.063, 0.031 and 0.016 M and these dilutions contained respectively 1.4,0.70, 0.35, 0.17, 0.085, 0.043, 0.021 and 0.011 M phosphate in the finalreaction mixture. Each tested ammonium-phosphate dilution also containeda small amount of 28 mM MOPS (3-(N-morpholino)propanesulfonic acid).

FIG. 2 shows the results of measuring the light signal in aluciferin-luciferase reaction mixture comprising different amounts ofethanolamine-phosphate (panel A) or ammonium-phosphate (panel B).Quantification of the reaction kinetics through surface area calculationunder the curves is shown in Table 4 (ethanolamine-phosphate dilutions)and Table 5 (for ammonium-phosphate dilutions).

As with Tris-phosphate, incrementing the ethanolamine-phosphateconcentration gives rise to a better light yield, but only up to 0.5 Methanolamine with 0.25 M phosphate. At 1.0 M ethanolamine with 0.5 Mphosphate the reaction results in a reduced light yield when compared tothe reaction mixture with 0.5 M ethanolamine with 0.25 M phosphate.Apparently, these concentrations are above optimum but neverthelessuseful.

Increasing ammonium-phosphate combinations up to 500 mM ammonium and 350mM of phosphate also causes increased light yield. At higherconcentrations the light yield decreases.

TABLE 4 Light yield of the luciferin-luciferase reaction as function ofethanolamine and phosphate concentration from FIG. 2A [Ethanolamine][Phosphate] Light Yield (M) (M) (10⁹counts) 1.0 0.5 99.0 0.5 0.25 124.80.25 0.125 115.4 0.125 0.063 96.2 0.063 0.031 70.0 0.031 0.016 59.20.016 0.008 49.6 0.008 0.004 41.6

TABLE 5 Light yield of the luciferin-luciferase reaction as function ofammonium and phosphate concentration from FIG. 2B [Ammonium] [Phosphate]Light Yield (M) (M) (10⁹counts) 2.0 1.4 44.6 1.0 0.7 80.7 0.5 0.35 113.10.25 0.175 88.9 0.125 0.088 83.2 0.063 0.043 69.3 0.031 0.021 43.7 0.0160.011 31.8

Example 3 Effect of Different Types of Reducing Agents

This example demonstrates the effect of various types of reducing agentsused to protect the reactive sulfhydryl group of luciferase fromoxidation. This can be important for a sustained activity of the enzyme.(DeLuca et al. Biochemistry 3:935 (1964); and Lee et al., Biochemistry8:130-136 (1969)). Various protective agents were compared against acontrol sample not containing a reducing agent in a luciferin-luciferasereaction comprising ammonium and phosphate ions (0.5 M Tris and 0.25 Mphosphate).

Stock solutions containing 100 mM of the following reducing agents wereprepared: sodium-dithionite (Na₂S₂O₄), dithiothreitol (DTT),Tris(2-carboxyethyl) phosphine (TCEP), sodium-thiosulfate (Na₂S₂O₃) andsodium-sulfite (Na₂SO₃). The TCEP stock solution was prepared bydissolving the hydrochloride salt (TCEP-HCl).

A 3.5 M Tris/1.8 M phosphate stock solution was diluted to aconcentration of 1.2 M Tris/0.62 M phosphate (or 1.11 M/0.56 M forNa₂S₂O₄). For each reducing agent a detection buffer was made bydiluting 100 μl of each reducing agent's stock (10 μl for Na₂S₂O₄) in800 μl Tris-phosphate buffer solution (890 μl for Na₂S₂O₄). Thedetection buffer was completed by adding 100 μl of a freshly madesolution with 30 mM ATP, 3 mM D-luciferin, pH 7.0. Luciferase samplesand light signal measurements were performed as described above. Thefinal concentration of the reducing agent in the reaction mixture was 5mM for DTT, TCEP, Na₂S₂O₃ and Na₂SO₃ and 0.5 mM for Na₂S₂O₄.

The time course of the light signal (FIG. 3) and the resulting lightyield of each reaction (Table 6) show that the presence of a reducingagent in the reaction mixture to protect the luciferase from oxidationresults in a large increase in light yield. Furthermore, the resultsdemonstrate the suitability of dithionite, thiosulfate, sulfite and TCEPas non-thiol reducing agents in a luciferin-luciferase assay.

TABLE 6 Light yield of the luciferin-luciferase reaction as function ofvarious reducing agents from FIG. 3 Reducing Light Yield Agent(10⁹counts) Na₂S₂O₄ 128.4 Na₂S₂O₃ 147.7 DTT 167.5 Na₂SO₃ 136.8 TCEP122.1 none 65.9

Example 4 Differential Effect of Different Ammonium/Phosphate IonConcentrations on the Luciferin-Luciferase Reaction in an ATP-DetectionVersus a Luciferase-Detection Assay

This example demonstrates that increasing the amounts of ammonium ionsand phosphate ions in the luciferin-luciferase reaction mixture has adifferent effect on an ATP-detection system than on aluciferase-detection system.

For this experiment 16 different reagent mixtures were prepared; 8mixtures (for the ATP assay) contained a large amount of luciferase, asmall amount of ATP and increasing Tris-phosphate concentrations and 8mixtures (for the luciferase assay) contained a large amount of ATP, asmall amount of luciferase and increasing amounts of Tris-phosphate.Final reaction mixture concentrations of each component are shown inTable 7. To reduce variances that might be introduced by smallconcentrations differences between the reagents, all reagents were madefrom the same stock solutions where possible.

A “luciferase sample” was prepared by reconstituting a lyophilizedluciferase control sample (PerkinElmer Inc.) in H₂O and diluting this inDulbecco's PBS with Ca/Mg (D′PBS⁺⁺) to a 1.0×10⁻⁷ g/l luciferasesolution.

An “ATP sample” was prepared by diluting a reconstituted lyophilized ATPstandard (PerkinElmer Inc.) in D′PBS⁺⁺ resulting in a 1.0×10⁻⁸ mM ATPsolution.

To measure the luminescence kinetics of the luciferin-luciferasereaction when assaying for ATP, 100 μl of each Tris-phosphateconcentration reagent with luciferase was pipetted into a 96-wellsCulturPlate™ (PerkinElmer Inc.) and 100 μl of “ATP sample” was added.Likewise, in the same microplate the reaction kinetics when assaying forluciferase were measured by pipetting 100 μl of each Tris-phosphateconcentration reagent with ATP, and adding 100 μl of “luciferasesample”.

The microplate was shaken for 10 seconds on a microplate-shaker andsealed with TopSeal-ATM (PerkinElmer Inc.). Thereafter the plate wasimmediately placed in a TopCount®-NTX to measure the emitted light. Theluminescent light signal was immediately measured, meaning about 1minute after mixing reagent and sample, and after 5 and 10 minutes.Hereafter the signal was measured repeatedly with 15 minute intervals toa total time of 420 minutes. Between the successive measurements themicroplate was kept constantly in the counter at 22° C. The emittedlight was quantified as counts per second (cps), count time 3 secondsper well.

FIG. 4A illustrates the effect of increasing Tris-phosphateconcentrations on the reaction kinetics in the ATP detection setting,and FIG. 4B shows the effect for the luciferase detection.

To further illustrate the difference between the ATP and the luciferasedetection systems, the total luminescence of each curve, i.e. theintegral under that curve between 1 and 420 minutes and expressed in 106counts, was calculated and plotted in FIG. 5 as a function of thephosphate concentration.

FIG. 6 shows the relationship between phosphate concentration and thehalf-life of the luminescence, i.e. the time needed for the luminescenceto decay to half its initial value (in minutes), for an ATP and aluciferase assay. Initial luminescence was assessed after 10 minutes ofincubation.

The ATP- and luciferase-detection assays clearly perform differentlywith respect to the increasing ammonium/phosphate ions in the reactionmixture. In an ATP assay, wherein the ratio luciferase-ATP is shiftedtowards the enzyme, higher Tris-phosphate ion concentrations inhibittotal luminescence. Whereas in a luciferase assay, wherein theluciferase-ATP ratio is shifted towards ATP, the presence of up to 100mM Tris-phosphate enhances total luminescence.

TABLE 7 Concentration of each component in 200 μl reaction mixture perwell for the ATP- or luciferase-detection assay. Concentration ofcomponents in final reaction mixture ATP assay Luciferase assay (8different reaction mixtures) (8 different reaction mixtures) ChemicalConcentration Concentration Trehalose 2.5 g/l 2.5 g/l HEPES 5 mM 5 mMBSA 0.5 g/l 0.5 g/l Na₂S₂O₃ 1.5 mM 1.5 mM TrisPO₄ ³⁻

NaCl 69 mM 69 mM CaCl₂ 0.45 mM 0.45 mM MgCl₂ 0.25 mM 0.25 mM KCl 1.3 mM1.3 mM D-luciferin 0.5 mM 0.5 mM ATP 5.0 × 10⁻⁹ M 1.5 × 10⁻³ MLuciferase 5.0 mg/l 5.0 × 10⁻⁵ mg/l pH 7.0 pH 7.0 *Corrected forphosphate ions present in PBS⁺⁺ used for sample preparation

1. A method of detecting luciferase activity in a sample, comprisingincubating the sample in the presence of luciferin and ATP and abivalent cation to allow the generation of a light signal, wherein thetotal light yield of said light signal is enhanced by performing theincubation in a reaction mixture comprising ammonium ions and at least20 mM phosphate ions, and measuring the light signal.
 2. Methodaccording to claim 1, wherein said phosphate concentration is more than60 mM.
 3. Method according to claim 1, wherein the phosphate ions areprovided by H₃PO₄, H₂PO₄ ⁻, or HPO₄ ²⁻, PO₄ ³⁻ or a combination thereof.4. Method according to claim 1, wherein said ammonium ion is provided byan ammonium salt, an amine or a zwitterionic compound, or a combinationthereof.
 5. Method according to claim 1, wherein said ammonium ion isprovided by an amine-based buffer, preferably Tris(tris(hydroxymethyl)aminomethane) or Bis-Tris(bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane).
 6. Methodaccording to claim 4, wherein said amine is a primary or secondaryamine, preferably an amine selected from the group consisting ofmonoethanolamine, diethanolamine, monoethylamine and diethylamine. 7.Method according to claim 1, wherein the ammonium ion concentration isat least 20 mM, preferably at least 60 mM, more preferably more than 100mM.
 8. Method according to claim 1, wherein the reaction mixture pHranges between 5 and
 9. 9. Method according to claim 1, wherein saidincubation is performed in the absence of exogenously added orsupplementary thiol-containing compound, such as DTT, glutathione orCoA, preferably wherein the incubation is performed in the absence ofDTT.
 10. Method according to claim 1, wherein said reaction mixturefurthermore comprises a reducing agent.
 11. Method according to claim10, wherein said reducing agent is a non-thiol compound, preferably acompound selected from the group consisting of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), thiosulfate, sulfite anddithionite. 12-17. (canceled)